1. Field of the Invention
This invention relates to methods and apparatus for performing microanalytic analyses and procedures. In particular, the present invention provides devices and methods for the performance of miniaturized cell based assays. These assays may be performed for a variety of purposes, including but not limited to screening of drug candidate compounds, life sciences research, and clinical and molecular diagnostics.
2. Background of the Related Art
Recent developments in a variety of investigational and research fields have created a need for improved methods and apparatus for performing analytical, particularly bioanalytical assays at microscale (i.e., in volumes of less than 100 xcexcL). In the field of pharmaceuticals, an increasing number of potential drug candidates require assessment of their biological function. As an example, the field of combinatorial chemistry combines various structural sub-units with differing chemical affinities or configurations into molecules; in theory, a new molecule having potentially unique biochemical properties can be created for each permutation of the sub-units. In this way, large libraries of compounds may be synthesized from relatively small numbers of constituents, each such compound being a potential drug lead compound of usually unknown biological activity and potency. Similarly, increasingly large numbers of targets for these putative therapeutic compounds are being discovered, many as a result of the growing information derived from such large-scale biological research as the sequencing of the human genome.
As the first phase of drug discovery, compounds that represent potential drugs are screened against targets in a process known as High Throughput Screening (HTS) or ultra-High Throughput Screening (uHTS). An advantage of these screening methods is that they usually consist of simple solution phase biochemical assays that can be performed quickly and with small amounts of expensive compounds and reagents. However, a significant drawback to HTS is that the targets do not provide a functional assessment of compounds"" effects on the complex biochemical pathways inherent in the normal and abnormal (mutant or disease-state) functioning of cells, tissues, organs, and organisms. As a result, compounds that have shown biochemical activity of interest in initial screens are usually put through cell-based screens, in which the affect of the compounds on cellular function is independently assayed.
There are a wide range of assays that may be performed using living cells. Assays that involve the use of living cells include gene expression, in which levels of transcription in response to a drug candidate are monitored; cell permeability assays, in which the ability of drugs to traverse membranes of cells is monitored; and functional assays designed to investigate both macroscopic effects, such as cell viability, as well as biochemical effects and products produced in and by the cells as a result of treatment with the drug lead compound.
These assays include cytotoxicity and cell proliferation to measure the viability of a population of cells, often in the presence of a putative therapeutic compound (drug candidate). A variety of methods have been developed for this purpose. These include the use of tetrazolium salts, in which mitochondria in living cells use dehydrogenases to reduce tetrazolium salts to colored formazan salts. Soluble or insoluble precipitates may be formed, depending on the nature of the tetrazolium salt used. A typical assay procedure is to culture the cells, add a solution of tetrazolium salt, phenazine methosulfate and DPBS, incubate, and determine absorbance at 490 nm. The absorbance measured is larger for viable cell populations that have metabolized the salt. Another such assay uses alamarBlue, which uses a fluorometric/colorimetric growth indicator that is reduced to a membrane-soluble, red, fluorescent form by the products of metabolic activity. A variety of other indicators are either taken up by living cells, dead cells, or both; for example, neutral red is taken up only by live cells, while trypan blue is excluded by live cells. Dyes that bind to or intercalate with DNA can be used to visualize or quantitate the number of live or dead cells, since DNA synthesis only occurs in living cells.
Another important class of cell based assays in reporter gene assays. These assays are used to study the control of gene transcription. They can also be used as a secondary detection method for a number of other molecules present in or acting on a cell. Pharmaceutical companies and others involved in drug development commonly utilize reporter gene assays to determine the effects of their compounds on transcription of specific genes whose promoter sequences are known. For example, the production of proteins associated with a condition of interest can be quantified by using a reporter gene operatively linked to the promoter of the gene encoding the protein. The method employed in reporter gene assays varies with the type of reporter gene used and the application. Initially, the promoter from the gene of interest in operable combination with the reporter gene is inserted into a commercially available plasmid comprising an antibiotic resistance gene, which is then transfected into the cells. Cells that have been successfully transfected can be selected by addition of the antibiotic, thereby eliminating the cells that have not been successfully transduced with the plasmid. When studying gene transcription, the cells are subsequently plated, compound(s) to be tested are introduced, and the assay for the reporter protein is conducted. These assays range from extremely simple to complex, with reporter proteins ranging from enzymes to hormones and photoproteins. Typically, enzymes are assayed using rate assays, hormones are detected using immunoassays, and photoproteins (e.g., green fluorescent protein, aequorin) are imaged optically.
Cell permeability assays measure the transport of compounds across cells. The commonly-used example is the CaCo-2 cell line derived from human intestinal endothelial cells. When grown to confluency over a porous membrane, these cells form a xe2x80x9cbiologically activexe2x80x9d filter: Transport of compound through the cell layer is accepted in the art to be correlated with absorbsion by the digestive system.
The compounds available for such cell-based testing have increased dramatically in recent years. In the decade from 1985 to 1995, drug library development through methods such as combinatorial chemistry and the discovery of new targets have created an explosive growth in both the number of compounds with promising biochemical properties. In order to effectively assay these xe2x80x9chitsxe2x80x9d using cell-based assays, an equivalent system of high throughput screening for such cell-based assays is needed.
To achieve the primary need of high throughput for cell based assays, a number of secondary features are desirable. First, it is advantageous to have a high degree of process automation, such as fluid transfer, cell plating and washing, and detection. It is also advantageous for the processes to be integrated so as to require a minimum of human intervention. Compound consumption (non-specific adsorption onto the materials comprising the assay apparatus) must be minimized, in order to prevent depletion of rare and/or expensive components of the compound libraries. This is most readily addressed through miniaturization of assays from their current scale of hundreds of microliters to ten microliters or less. A goal in the art is to provide automated, integrated and miniaturized apparatus for performing assays that are reliable and produce results consistent with the results produced by current, more laborious, expensive and time-consuming assays.
In addition to these advantages, miniaturization itself can confer performance advantages. At short length scales, diffusionally-limited mixing is rapid and can be exploited to create sensitive assays (Brody et al., 1996, Biophysical J. 71: 3430-3431). Because fluid flow in miniaturized pressure-driven systems is laminar, rather than turbulent, processes such as washing and fluid replacement are well-controlled. Miniaturized, most advantageously microfabricated systems also enable assays that rely on a large ratio of surface area to volume, such chromatographic assays generally and assays that require binding to a surface.
Miniaturization has led to the creation of 384-well and 1536-well microtiter plates for total reaction volumes of between 0.015 and 0.1 mL. However, a number of problems arise when miniaturizing standard plate technology, especially for use in conjunction with cells. First, because the total volumes are smaller and the plates are open to the environment, evaporation of fluid during the course of an assay can compromise results; this is especially problematic for cell based assays that may require incubation at elevated temperatures for up to several days. Another drawback of open plates is the existence of the meniscus of fluid in the well. Meniscuses of varying configurations (due, for example to imperfections in the plate or differences in contact angle and surface tension) can distort the optical signals used to interrogate the samples. As the strength of the optical signals decreases with decreasing assay volume, correction for background distortions becomes more difficult. Finally, optical scanning systems for high-density plates are often complex and expensive. Methods that minimize evaporation, provide a more uniform optical pathway, and provide simpler detection schemes are desirable.
Highly accurate pipetting technologies have been developed to deliver fluids in precisely metered quantities to these plates. Most of these fluid-delivery methods for low volumes (below approximately 0.5 xcexcL) rely on expensive piezoelectric pipetting heads that are complex and difficult to combine or xe2x80x9cgangxe2x80x9d into large numbers of independent pipettors so that many wells may be addressed independently. As a result, fluid delivery is either completely or partially serial (i.e., a single micropipettor, or a small number of parallel delivery systems used repeatedly to address the entire plate). Serial pipetting defeats the aim of parallelism by increasing the amount of time required to address the plate. Methods that reduce the number and precision of fluid transfer steps are therefore needed.
Attempts to produce microfabricated devices for performing cell-based assays have been reported in the art. For example, International Patent Application WO98/028623, published 2, Jul. 1998 by several of the instant inventors, discloses a microfluidics platform for detecting particulates in a fluid, specifically including cells.
A microfabricated device explicitly for the performance of cell based assays in a centrifugal format has been disclosed in International Patent Application WO 99/55827, published November 1999. The operative principles of this device include the use of hydrophobic coatings along a radial channel punctuated by cell culturing chambers and optical cuvettes. However, this device cannot perform distinct assays on subpopulations of the cells cultured on the device. By providing only a single entry to a multiplicity of cell culturing chambers, all chambers are exposed to the same solutions, such as cell suspension, cell culture medium, test compounds and any reagents used for detection of the effects of these compounds. Furthermore, the format disclosed in WO 99/55827 relies on the manufactured surface of the microplatform to provide the support for cell attachment and proliferation, or the use of carrier beads. This may not be adequate for all cell types of interest. Finally, no provision is made for selectively trapping and incubating certain cells or cell types rather than others. In applications such as diagnostics, in which a variety of cells may be present in a biological sample such as blood, means for separating cells based on type or other features may be required.
Thus, there is a need in the art for improved micromanipulation apparatus and methods for performing cell based assays more rapidly and economically using less biological sample material. Relevant to this need in the art, some of the present inventors have developed a microsystem platform and a micromanipulation device to manipulate said platform by rotation, thereby utilizing the centripetal forces resulting from rotation of the platform to motivate fluid movement through microchannels embedded in the microplatform, as disclosed in co-owned U.S. Pat. No. 6,063,589, issued May 16, 2000, and co-owned and co-pending patent application U.S. Ser. Nos. 08/761,063, filed Dec. 5, 1996 now abandoned; 08/768,990, filed Dec. 18, 1996 now U.S. Pat. No. 6,319,469; 08/910,726, filed Aug. 12, 1997 now U.S. Pat. No. 6,143,248; 08/995,056, filed Dec. 19, 1997 now U.S. Pat. No. 6,143,243; and 09/315,114, filed May 19, 1999 now U.S. Pat. No. 6,632,399, the disclosures of each of which are explicitly incorporated by reference herein.
The invention disclosed herein relates to microfluidic devices for performing cell based assays for a variety of applications such as life sciences, diagnostics and drug screening. In particular, these devices have been developed to carry out various steps common to many cell-based assays. The devices comprise an entry port or other means for adding cellular suspensions, most preferably in vitro cell cultures, into the devices of the invention. Surfaces and supports comprising the devices have been adapted or treated to permit cell attachment and growth to occur on appropriate surfaces and supports in the devices, while alternatively other surfaces or components of the devices have been fabricated or treated to inhibit cell attachment and growth. The components of the devices are arranged to permit cell growth on the surface of the device, including such attendant process as exchange of growth media, exchange of gases like carbon dioxide naturally respired during growth, and incubation at temperatures appropriate for cell culture. The devices of the invention are produced to facilitate distribution of test solutions to the cells cultured within the device, said solutions preferably carrying test compounds or other reagents. Finally, the components of the devices of the invention are provided so that metabolites, secretions, or excretions from cells on the device can be detected, either directly or through reaction with appropriate reagents. Another preferred form of detection provided is the direct visualization and imaging of cells.
This invention provides microsystems platforms as disclosed in co-owned U.S. Pat. No. 6,063,589, issued May 16, 2000, and co-owned and co-pending patent application U.S. Ser. Nos. 08/61,063, filed Dec. 5, 1996; 08/768,990, filed Dec. 18, 1996; 08/910,726, filed Aug. 12, 1997; 08/995,056, filed Dec. 19, 1997; and 09/315,114, filed May 19, 1999, the disclosures of each of which are explicitly incorporated by reference herein, adapted to permit cell attachment and growth on the surface thereof, most preferably in specific components such as cell growth chambers as described herein. Additional microfluidics components that facilitate the performance of cell based assays are also provided, as described in more detail herein.
The invention provides apparatus and methods for performing microscale processes on a microplatform, whereby fluid is moved on the platform in defined channels motivated by centripetal force arising from rotation of the platform. The first element of the apparatus of the invention is a microplatform that is a rotatable structure, most preferably a disk, the disk comprising fluid (sample) inlet ports, fluidic microchannels, reagent reservoirs, cell growth or aggregation chambers, detection chambers and sample outlet ports, generically termed xe2x80x9cmicrofluidic structures,xe2x80x9d and also comprise heating elements that make up a portion of the surface area of the platform for heating fluids contained therein to temperatures greater than ambient temperature. In preferred embodiments, said heating elements are positioned on the disk in sufficient proximity to the cell growth or aggregation chambers to allow cell growth in said chambers on the disk surface without inhibiting cell growth or otherwise compromising the viability of growing cells. The disk is rotated at speeds from about 1-30,000 rpm for generating centripetal acceleration that enables fluid movement through the microfluidic structures of the platform. The disks of the invention also preferably comprise air outlet ports and air displacement channels. The air outlet ports and in particular the air displacement ports provide a means for fluids to displace air, thus ensuring uninhibited movement of fluids on the disk. These air outlet ports also permit gas exchange between cell growth or aggregation chambers and the atmosphere, to allow the culture media to be oxygenated and to eliminate gaseous waste such as carbon monoxide. Specific sites on the disk also preferably comprise elements that allow fluids to be analyzed, as well as detectors for each of these effectors. Alternatively, some or all of these elements can be contained on a second disk that is placed in optical or direct physical contact, most preferably thermal contact, with the first platform disk.
The discs of this invention have several advantages over those that exist in the centrifugal analyzer art. Foremost is the fact that flow is laminar due to the small dimensions of the fluid channels; this allows for better control of processes such as mixing and washing. To this are added the already described advantages of miniaturization, as described in more detail above.
The second element of the invention is a micromanipulation device that is a disk player/reader device that controls the function of the disk. This device comprises mechanisms and motors that enable the disk to be loaded and rotated. In addition, the device provides means for a user to operate the Microsystems in the disk and access and analyze data, preferably using a keypad and computer display. The micromanipulation device also advantageous provides means for actuation of on-disc elements, such active valves; the application and control of heat to the disc for purposes of chemical or biological incubation; and means for adding fluids to and removing fluids from the discs. In preferred embodiments, the apparatus also comprises means for insulating the platforms of the invention from the environment, so that cells growing on the disc can be maintained at the proper temperature, oxygen tension, acidity, humidity levels, and other parameters understood by those with skill in the cell culture arts.
The invention specifically provides Microsystems platforms comprising microfluidics components contained in one or a multiplicity of platform layers that are fluidly connected to permit transfer, mixing and assay performance on the sealed surface of the platform. The platforms preferably comprise one or more entry ports through which cell suspensions may be added in volumes ranging from about 1 nL to about 1 mL. The platforms preferably comprise one or more reagent reservoirs containing a sufficient volume, preferably from about 1 nL to about 1 mL, of a reagent solution for a multiplicity of individual assays. The reagent reservoirs are fluidly connected by microchannels to one or preferably a multiplicity of cell incubation chambers. These cell incubation chambers are preferably equipped with a surface that has been constructed or specifically adapted for attachment and growth of cells, and may also be sealed with selectively-permeable membranes for which allow passage of gases in and out of the chambers from the exterior environment. Cell incubation chambers may be equipped with devices for capturing cells passed through the chambers. Additionally, the cell incubation chambers may be fluidly connected to detection chambers and waste chambers. In some preferred embodiments, the platform comprises a multiplicity mixing channels and reservoirs for the mixing of reagents in various ratios and for the creation of dilution series for performing cell-based assays of drugs and other compounds.
In the use of the platforms of the invention, fluids (including cell suspensions and reagents) are added to the platform when the platform is at rest. Thereafter, rotation of the platform on a simple motor motivates fluid movement through microchannels for various processing steps. In preferred embodiments, the platforms of the invention permit the use of a detector, most preferably an optical detector, for detecting the products of an assay, most preferably a biochemical assay, whereby the assay reaction chambers comprise optical cuvettes, preferably positioned at the outer edge of the platform, and most preferably wherein the platform is scanned past a fixed detector through the action of the rotary motor. In other embodiments, the platforms permit the use of optical imaging systems for the direct visualization of cells that have attached to support surfaces within the platform Because the platforms of the invention are most preferably constructed using microfabrication techniques as described more fully below, the volumes of fluids used may be made arbitrarily small as long as the detectors used have sufficient sensitivity.
The present invention solves problems in the current art through the use of a microfluidic disc in which centripetal acceleration is used to move fluids. It is an advantage of the microfluidics platforms of the present invention that the fluid-containing components are constructed to contain small volumes, thus reducing reagent costs, reaction times and the amount of biological material required to perform an assay. It is also an advantage that the fluid-containing components are sealed, thus eliminating experimental error due to differential evaporation of different fluids and the resulting changes in reagent concentration, as well as reducing the risk of contamination, either of the cell culture or the operator. Because the microfluidic devices of the invention are completely enclosed, both evaporation and optical distortion are reduced to negligible levels. The platforms of the invention also advantageously permit xe2x80x9cpassivexe2x80x9d mixing and valving, i.e., mixing and valving are performed as a consequence of the structural arrangements of the components on the platforms (such as shape, length, position on the platform surface relative to the axis of rotation, and surface properties of the interior surfaces of the components, such as wettability as discussed below), and the dynamics of platform rotation (speed, acceleration, direction and change-of-direction), and permit control of assay timing and reagent delivery.
In alternative embodiments of the platforms of the invention, metering structures as disclosed in co-owned U.S. Pat. No. 6,063,589, issued May 16, 2000 and incorporated by reference herein, are used to distribute aliquots of reagent to each of a multiplicity of mixing structures, each mixing structure being fluidly connected to one of a multiplicity of sample reservoirs, thereby permitting parallel processing and mixing of the samples with a common reagent. This reduces the need for automated reagent distribution mechanisms, reduces the amount of time required for reagent dispensing (that can be performed in parallel with distribution of reagent to a multiplicity of reaction chambers), and permits delivery of small (nL-to-xcexcL) volumes without using externally-applied electromotive means. It also enables the performance of multiplexed assays, in which cell populations may be divided and the microfluidics of the device used to perform a variety of assays on different sub-populations in parallel, on one population serially, or on a single population simultaneously.
A further advantage of the platforms of the invention is the use of elements that can serve to selectively capture cells based on properties such as size (though the use of porous filters) or type (through the use of immunochemical methods, as disclosed in co-owned U.S. Pat. No. 6,143,247, issued 7, Nov. 2000 and International Application Publication No. WO98/28623, published 2, Jul. 1999, the teachings of each of which are explicitly incorporated by reference herein).
The assembly of a multiplicity of cell incubation chambers on the platforms of the invention also permits simplified detectors to be used, whereby each individual reaction chamber can be scanned using mechanisms well-developed in the art for use with, for example, CD-ROM technology. Finally, the platforms of the invention are advantageously provided with sample and reagent entry ports for filling with samples and reagents, respectively, that can be adapted to liquid delivery means known in the art (such as micropipettors).
The platforms of the invention reduce the demands on automation in at least three ways. First, the need for precise metering of delivered fluids is relaxed through the use of on-disc metering structures, as described more fully in co-owned U.S. Pat. No. 6,063,589, issued May 16, 2000, and co-owned and co-pending patent application U.S. Ser. Nos. 08/761,063, filed Dec. 5, 1996; 08/768,990, filed Dec. 18, 1996; 08/910,726, filed Aug. 12, 1997; 08/995,056, filed Dec. 19, 1997; and 09/315,114, filed May 19, 1999, the disclosures of each of which are explicitly incorporated by reference herein. By loading imprecise volumes, slightly in excess of those needed for the assay, and allowing the rotation of the disc and use of appropriate microfluidic structures to meter the fluids, much simpler (and less expensive) fluid delivery technology may be employed than is the conventionally required for high-density microtitre plate assays.
Second, the total number of fluid xe2x80x9cdeliveryxe2x80x9d events on the microfluidic platform is reduced relative to conventional assay devices such as microtiter plates. By using microfluidic structures that sub-divide and aliquot common reagents (such as reagent solutions, buffers, and enzyme substrates) used in all assays performed on the platform, the number of manual or automated pipetting steps are reduced by at least half (depending on the complexity of the assay). Examples of these structures have been disclosed in co-owned U.S. Pat. No. 6,063,589, issued May 16, 2000, and incorporated by reference herein. In some examples shown explicitly herein, the microfluidic structures of the platform may be used to create, for example, multiple mixtures of reagents with different mixing ratios for application to cultured cells. These structures provide automation, for example, for serial dilution assays, a laborious process when performed conventionally. This process is replaced by xe2x80x9cparallel dilutionxe2x80x9d on the platforms of the invention.
Finally, the invention also provides on-platform means for adding incubation media, washing cell incubation chambers, and media replacement. These features also reduce manipulation of the assay device by, for example, robotic washing stations, as well as providing controlled and integrated fluid processing.
Certain preferred embodiments of the apparatus of the invention are described in greater detail in the following sections of this application and in the drawings.